This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While vertebrate eye development and functional differentiation is initiated in mid gestation, it is of note that the retina is perhaps the last organ to complete functional development. Most retinal differentiation occurs postnatally, and in the mouse retinal development is not completed until two weeks after birth. Thus, developmental defects in the retina are not evident functionally until significantly after birth. The retinal pigmenl epithelium (RPE) cells are highly polarized and they become tightly associated with the developing retinal photoreceptor cells. Once the retina becomes functional, the RPE is essential for phagocytic clearance of the spent photoreceptor distal outer segment (OS) tips;this function is essential to maintain photoreceptor homeostasis. The RPE develops well before the retina, but developmental defects in the RPE do not become evident until the postnatal period when the retina becomes functional. Developmental defects in RPE phagocytosis of the OS leads to photoreceptor death and the postnatal disease retinitis pigmentosa (RP). The role of the RPE in rod OS turnover has been extensively studied, due to the availability of the Royal College of Surgeons (RCS) rat model, which displays an RP phenotype. The RPE cells in these rats carry an inherited defect in rod OS phagocytosis and the mutant gene is the MerTK receptor. The MerTK receptor tyrosine kinase is essential for RPE phagocytosis. MerTK null mutation causes photoreceptor degeneration not only in the RCS rat, but also in MerTK gene knockout mice and human RP patients. In addition, the MerTK mutation leads to general developmental defects in NK cell differentiation, spermatogenesis, and macrophage phagocytosis. To understand the molecular role of MerTK in phagocytosis in these developmental process, we performed gene expression profiling to identify downstream targets of MerTK. One gene known as PTTG was ectopically expressed with MerTK mutation in RPE and macrophages. Interestingly, PTTG regulates cytoskeletal changes in dividing cells, PTTG stabilizes cytoskeletal structures such as the spindle apparatus, which separates sister chromatids during mitosis. However, PTTG must be degraded for this cytoskeletal structure to be disassembled at the end of mitotic metaphase. Thus, PTTG is required for cytoskeletal changes to occur in the cell. We have now crossed the MerTK mutant mice into a PTTG heterozygous background to reduce ectopic expression of PTTG, and we have found that photoreceptor loss in the MerTK mutant is largely prevented. These results point toward ectopic expression of PTTG as a critical factor in the RPE defects in the MerTK mutant. We hypothesize that ectopic expression of PTTG in the MerTK mutant RPE prevents the cytoskeletal rearrangements required to initiate phagocytosis (in a fashion analogous to that seen in cytoskeletal rearrangement defects seen in mitosis when PTTG becomes overexpressed). We now focus this proposal on investigation of the potential role for PTTG ectopic expression in the MerTK mutant phenotypes, and how the PTTG affect MerTK-mediated phagocytosis. Specific Aim 1. Examination of developmental and circadian regulation of PTTG expression and its role in phagocytosis of photoreceptor OS in the MerTK-/- RPE cells in vivo. In this Specific Aim, we will investigate the developmental expression pattern and circadian regulation of PTTG in RPE cells at both mRNA and protein level. We will study the photoreceptor degeneration time course and determine if phagocytosis is restored in MerTK-/-PTTG+/- mice. Specific Aim 2. Investigate whether in vitro ectopic expression of PTTG leads to defects in RPE phagocytosis. We will ectopically PTTG in normal RPE cells to examine if it will cause RPE cell phagocytic defects. Conversely, we will knockdown PTTG expression by RNA interference in the RPE cells with MerTK mutation and investigate whether decreased PTTG expression is sufficient to restore phagocytosis. Specific Aim 3. Functional studies of the MerTK-/- and MerTK-/-PTTG+/- retina by visual test and ERG analysis. We will perform electrophysiology and functional vision measurements on the MerTK-/-PTTG+/- mice. These physiological measurements will be correlated with a detailed morphological analysis of retinal markers and ultrastructual morphology.